Acta Vet. Brno 2017, 86: 167-174

Detection of selected antibiotic resistance genes using multiplex PCR assay in mastitis pathogens in the Czech Republic

Vladimir Pyatov, Irena Vrtková, Aleš Knoll

Mendel University in Brno, Faculty of AgriSciences, Department of Animal Morphology, Physiology and Genetics and CEITEC MENDELU, Brno, Czech Republic

Received November 21, 2016
Accepted May 31, 2017

The aim of this research was to develop multiplex polymerase chain reaction assays for the detection of aminoglycoside (strA, strB), sulphonamide (sulI, sulII), tetracycline (tetA, tetB, tetK, tetM, tetO), macrolide and lincosamide (msrA, ermA, ermB, ermC, mefA/E) genes of resistance in mastitis pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae). Applying the established assays, we investigated the distribution of antibiotic resistance genes in the above mentioned species isolated from milk samples in the Czech Republic. Each assay consisted of seven pairs of primers. Six of them amplified fragments of antibiotic resistance genes and one pair a fragment of a species specific gene. Polymerase chain reaction conditions were optimized to amplify seven gene fragments simultaneously in one reaction. In total, 249 isolates were used, among which 111 were positive for E. coli, 52 for S. aureus and 86 for Streptococcus spp. The majority (60.2%) of bacteria carried at least one antibiotic resistance gene and 44.6% were multidrug-resistant. The designed multiplex polymerase chain reaction assays may be applied as diagnostic method to replace or complement standard techniques of antibiotic susceptibility testing in the mentioned pathogens.


The study was supported by the project of the Ministry of Agriculture of the Czech Republic QJ1210301 and was carried out under the project CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Programme II.


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